Monoclonal antibodies have provided new therapies for the treatment of various disorders including cancer, immunological and neurological disorders and also infectious diseases. Newsome, B. W. et al., Br J Clin Pharmacol 66(1):6-19 (2008); Chames, P., et al., Br J Pharmacol 157(2):220-33 (2009); Dimitrov, D. S. et al., Methods Mol Biol 525:1-27, xiii (2009). These therapies have been successful, at least in part, because of the robust and strong interaction with target proteins and the singular specificity that monoclonal antibodies provide. The relatively long half-life and stability of monoclonal antibodies in vivo allow for desirable dosing regimens and cell-mediated toxicity can be engaged by the Fc region of the antibody (Tabrizi, M. A., et al., Drug Discov Today 11(1-2):81-8 [2006]). In certain instances, therapeutic antibodies have been used to block cellular signals by binding to and neutralizing important functional regions of secreted and cell-surface proteins. Such basic properties of monoclonal antibodies are currently being used to design molecular therapies with different mechanisms of action compared to traditional antibodies (Dimitrov, D. S. et al., Methods Mol Biol 525:1-27, xiii [2009]). Certain such technologies are currently in clinical development and show signs of promise (Chames, P., et al., Br J Pharmacol 157(2):220-33 [2009]).
For example, one approach involves cell-specific targeting using antibodies to deliver cytotoxic drugs to tumors. Carter, P. J. et al., Cancer J 14(3):154-69 (2008); Junutula, J. R., et al., Nat Biotechnol 26(8):925-32 (2008); Senter, P. D., Curr Opin Chem Biol 13(3):235-44 (2009). In this case, monoclonal antibody specificity directs the cytotoxic molecules to target cells thereby concentrating the high toxicity of the cytotoxic moiety where it is needed while minimizing the impact to nontarget cells. Such antibody-drug conjugates allow for increasing the potency in killing tumor cells while maintaining a window of dosing that minimizes off-target toxicity.
Another example is the delivery of functional complexes such as nanoparticles containing agents such as siRNAs and that include monoclonal antibodies on the surface of the particles for targeting. Schiffelers, R. M., et al., Nucleic Acids Res 32(19):e149 (2004); Vornlocher, H. P., Trends Mol Med 12(1):1-3 (2006); Davis, M. E., Mol Pharm 6(3):659-68 (2009).
Yet another approach uses the bivalent structure of antibodies to construct bispecific molecules that bind to two targets simultaneously (Fischer, N. et al., Pathobiology 74(1):3-14 [2007]). Bispecific antibodies offer opportunities for increasing specificity, broadening potency, and utilizing novel mechanisms of action that cannot be achieved with a traditional monoclonal antibody. Drakeman, D. L., Expert Opin Investig Drugs 6(9):1169-78 (1997); Kontermann, R. E., Acta Pharmacol Sin 26(1):1-9 (2005); Marvin, J. S. et al., Acta Pharmacol Sin 26(6):649-58 (2005); Marvin, J. S., et al., Curr Opin Drug Discov Devel 9(2):184-93 (2006); Shen, J., et al., J Biol Chem 281(16):10706-14 (2006); Chames, P. et al., Curr Opin Drug Discov Devel 12(2):276-83 (2009). Cross-linking two different receptors using a bispecific antibody to inhibit a singling pathway has shown utility in a number of applications. In one example, a cell-surface tyrosine phosphatase was recruited into an IgE receptor complex to decrease activity of the phosphorylated IgE receptor (Jackman, et al., J. Biol. Chem. 285:20850-20859 (2010)). This approach was more effective than blocking the ligand binding site because inhibition of signaling by the bispecific antibody occurred even in the presence of high concentrations of ligand. Id.
The use of bispecific antibodies to recruit cytotoxic T-cells has also shown clinical opportunities where T-cell activation was achieved in proximity to tumor cells by the bispecific antibody binding receptors simultaneously on the two different cell types. Bargou, R., E., et al., Science 321(5891):974-7 (2008); Shekhar, C., Chem Biol 15(9): 877-8 (2008); Baeucrle, P. A., et al., Cancer Res 69(12):4941-4 (2009). In one approach, a bispecific antibody having one arm which bound FcγRIII and another which bound to the HER2 receptor was developed for therapy of ovarian and breast tumors that overexpress the HER2 antigen. (Hseih-Ma et al. Cancer Research 52:6832-6839 [1992] and Weiner et al. Cancer Research 53:94-100 [1993]). Bispecific antibodies can also mediate killing by T cells. Typically, the bispecific antibodies link the CD3 complex on T cells to a tumor-associated antigen. A fully humanized F(ab)2 bispecific antibody consisting of anti-CD3 linked to anti-p185HER2 was used to target T cells to kill tumor cells overexpressing the HER2 receptor. Shalaby et al., J. Exp. Med. 175(1):217 (1992). Bispecific antibodies have been tested in several early phase clinical trials with encouraging results. In one trial, 12 patients with lung, ovarian or breast cancer were treated with infusions of activated T-lymphocytes targeted with an anti-CD3/anti-tumor (MOC31) bispecific antibody. deLeij et al. Bispecific Antibodies and Targeted Cellular Cytotoxicity, Romet-Lemonne, Fanger and Segal Eds., Lienhart (1991) p. 249. The targeted cells induced considerable local lysis of tumor cells, a mild inflammatory reaction, but no toxic side effects or anti-mouse antibody responses.
In addition, bispecific antibodies may be used in the treatment of infectious diseases (e.g. for targeting of effector cells to virally infected cells such as HIV or influenza virus or protozoa such as Toxoplasma gondii), used to deliver immunotoxins to tumor cells, or target immune complexes to cell surface receptors. See, e.g., Fanger et al., Crit. Rev. Immunol. 12:101-124 (1992). For example, with respect to HIV infection, Berg et al., PNAS (USA) 88:4723-4727 (1991) made a bispecific antibody-immunoadhesin chimera which was derived from murine CD4-IgG. These workers constructed a tetrameric molecule having two arms. One arm was composed of CD4 fused with an antibody heavy-chain constant domain along with a CD4 fusion with an antibody light-chain constant domain. The other arm was composed of a complete heavy-chain of an anti-CD3 antibody along with a complete light-chain of the same antibody. By virtue of the CD4-IgG arm, this bispecific molecule binds to CD3 on the surface of cytotoxic T cells. The juxtaposition of the cytotoxic cells and HIV-infected cells results in specific killing of the latter cells.
A number of methods have been described for the synthesis of multispecific antibodies, including bispecific antibodies. Methods for the synthesis of divalent antibody fragments have been described in WO 99/64460. Many of these approaches, however, present a variety of problems. For example, difficulties with protein expression and large scale production, stability and in vivo half-life, folding and aggregation have all been reported. Morimoto, K., et al., J Immunol Methods 224(1-2):43-50 (1999); Kriangkum, J., et al., Biomol Eng 18(2):31-40 (2001); Segal, D. M. and B. J. Bast (2001). “Production of bispecific antibodies.” Curr Protoc Immunol Chapter 2:Unit 2 13; Graziano, R. F., et al., Methods Mol Biol 283:71-85 (2004); Kontermann, R. E., et al., Methods Mol Biol 248:227-42 (2004); Das, D., et al., Methods Mol Med 109:329-46 (2005); Fischer, N. et al., Pathobiology 74(1):3-14 (2007); Shen, J., et al., J Immunol Methods 318(1-2):65-74 (2007). In addition, many of these methods are cumbersome and time-consuming thus limiting the number and variety of molecules that can be constructed and screened for desired activities. The methods described herein address these problems and the methods, compositions, multispecific antibodies and antibody analogs described herein provide additional benefits.
All references cited herein, including patent applications and publications, are incorporated by reference in their entirety for any purpose.